Skipped Over: Tuning Natural Killer Cells Toward HIV Through Alternative Splicing

Alternative splicing is a mechanism used by cells to maximize the available genetic code by producing several potentially viable transcripts from a single multiexon gene. Although the process of gene splicing has been known for several decades, how splicing is regulated and how the splicing machinery could be coopted therapeutically are still largely underexplored. It is well established that the spliceosome complex is important in regulating gene splicing ¹ and several studies have suggested the role of splicing in cancers, ² but how these components are altered during situations of cellular stress, infections, or other disease states is largely unexplored. ^3,4

Recent study by Rotival et al. clearly demonstrates that treating human monocytes with several toll-like receptor agonists (proxies for viral, bacterial, or fungal infections) resulted in varying gene isoform expression profiles, including the production of putatively dysfunctional isoforms. ⁵ It has also been shown that macrophage function is altered in the context of Mycobacterium tuberculosis infection through modulation of the splicing patterns of several genes, diminishing the ability of macrophages from clearing infection. ⁶ Viral infection with, but not limited to, dengue, ⁷ ZIKV, ⁸ poliovirus, ⁹ and HIV ¹⁰ has also been shown to result in changes to the splicing patterns of host cells.

Although obviously not the only cause, it is possible that modulation of the host splicing patterns contributes to the pathology of infection. ¹¹ For example, during HIV infection, trans-activator of transcription (TAT) seems to be associated with neurocognitive effects through modulation of the host splicing machinery. ¹⁰ Changes in alternative splicing are not limited to disease states, but also occur naturally during aging. Several studies have shown that in aged patient cohorts, there are fewer gene isoforms produced in immune cells, and this is correlated with weaker immune responses and poorer disease outcomes. ^12,13

Natural killer (NK) cells are potent antiviral and antitumor immune cells that play a crucial role in regulating HIV/SIV disease, particularly during acute infection. ¹⁴ Interestingly, chronic infection with HIV/SIV leads to a premature immune aging phenotype, ¹⁵ including a decline in NK cell functions, perhaps through exhaustion mechanisms that resemble those observed in T cells. ¹⁶ This is evident through NK cell acquisition of exhaustion markers such as Tim-3, or decreased expression of transcription factors such as Eomes, both of which when blocked (Tim-3) or reversed (through Eomes overexpression) result in the restoration of NK cell function. ¹⁶

Interestingly, genes encoding both Eomes and Tim-3 have multiple isoforms that may be differentially regulated during disease. It is well established that infection and disease lead to immune modulation through release of various cytokines and mobilizing various cell types, yet a comprehensive undertaking looking at how infection or disease results in an altered isoform landscape does not currently exist. Previous study from the Poltorak laboratory has shown that genetic contribution of specific gene isoforms of the apoptosis regulator cellular FLICE (FADD-like IL-1-converting enzyme)-inhibitory protein (cFLIP) leads to improved outcomes toward septic shock in a mouse model and that this can be transferred to nonresistant mice through genetic modulation of a single gene isoform. ¹⁷ This study suggests that targeting gene isoform expression may be a viable therapeutic approach that may also be able to overcome issues arising from a complete knockdown of gene expression.

We posit that a similar approach can be employed in reversing the immunoregulatory effects after retroviral infection with HIV/SIV. Indeed analysis of data from our group in rhesus macaque NK cells ¹⁸ has identified several genes that exist as splice variants at preinfection time points, including elevated short cFLIP isoforms, and NKp46 and NKp30. As a test case and proof of concept for this perspective, we more closely evaluated our own data (Fig. 1A) as well as published data on NKp30 (Fig. 1B–F). Data from human uterine NK cells revealed that NKp30 isoform expression was modulated by exposure to different cytokines and that isoform expression seemed to also be associated with disparate functional properties. ¹⁹

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FIG. 1.

NKp30 isoform-specific characterization in rhesus macaque and human NK cells. Transcripts per million read counts showing NKp30 isoforms in NK cells from (A) naive rhesus macaques (GSE148290); reanalysis of publicly available human RNA-Seq cohorts from (B) heterogeneous patient samples (GSE60424); (C) samples stimulated with or without adenosine (GSE119705); (D) samples after withdrawal of (No) or stimulation with IL-2 (+IL-2) or IL-15 (+IL-15), assessing RNA from the polysomal fraction (Polysomal) or cytoplasmic fraction (Cyto)(GSE77808); (E) samples unstimulated (Unstim) or stimulated with PMA and ionomycin (+PMA) from normal or DOCK8 knockout (DOCK8 KO) KHYG1 NK cell line (GSE101467); (F) patient samples after injection with the trivalent inactivated influenza vaccine from preinfection (day 0) to day 7 postinoculation (GSE64655). In the rhesus (A), the 201aa isoform corresponds with (ENSMMUP00000011616.3) and the 220aa isoform corresponds with (ENSMMUP00000011617.4). In the human samples (B–F), the 165aa isoform corresponds with (ENSP00000365239.4), the 190aa isoform corresponds with (ENSP00000365240.3), the 177aa isoform corresponds with (ENSP00000365241.4), and the 201aa isoform corresponds with (ENSP00000342156.5). The isoform-specific analyses of rhesus macaque naive samples were done using salmon ³² and IsoformSwitchAnalyzeR, ³³ reanalysis of the human data was done using Grein. ²⁰ NK, natural killer.

Reanalysis of several publicly available data sets from Gene Expression Omnibus (GEO) using GREIN ²⁰ reveals that in human NK cells, isoform-specific expression of NKp30 isoforms varies in a heterogeneous population (GSE60424, Fig. 1B) with a preference for expression of a 177 amino acid variant (ENST00000376073.8). Isoform-specific NKp30 modulations also occur after ex vivo or in vitro stimulation (GSE77808, GSE101467, and GSE119705; Fig. 1C–E) or even after vaccination against influenza (GSE64655; Fig. 1F).

In our rhesus macaque data, we see expression of two NKp30 gene isoforms with a higher expression of a longer variant (220 amino acids, ENSMMUT00000012387.4) over a shorter variant (201 amino acids, ENSMMUT00000012386), Figure 1A. There is a 90% overlap between the predominant human and rhesus isoforms with the major differences in the signal peptide sequences. Whether or not these isoforms are functionally different or expression levels of these isoforms are altered during retroviral infection will require further investigation. NKp30 is just one example of many genes that are expressed as alternative transcripts; indeed, this deserves a more thorough investigation. In particular, it may be useful to assess the role that alternative splicing has on killer Ig-like receptor (KIR) selection in NK cells as described by Bruijnesteijn et al. ²¹ especially in the context of disease states given the role that KIR plays in MHC antigen recognition by NK cells.

Since NK cells themselves are not infected with HIV/SIV, global immune modulation by the virus may lead to changes in NK cell alternative gene splicing—which can result in altered NK cell responses. It has already been shown that NK cells utilize alternative splicing mechanisms to modulate HLA-C expression, possibly leading to varying degrees of self-recognition, ²² so it is possible that NK cells may also fine-tune antiviral responses through alternative splicing events. If we can understand the changes in splicing patterns, as well as identify key genes leading to NK cell dysfunction, we may be able to reverse the consequences of retroviral-induced immunosuppression observed during chronic HIV/SIV infection. Furthermore, NK cells can also generate memory-like immune responses against SIV epitopes. ²³ Since NK cells do not utilize RAG recombinase, it is possible that alternative splicing mechanisms may provide an opportunity to modify genes of other antigen recognition molecules in NK cells to provide epitope-specific responses.

Most RNA intervention programs are aimed at targeting the infection, disrupting the immune response through dampening of cytokine signaling, or inhibition of immunosuppressive molecules leading to enhanced immune responses. ²⁴ Sometimes, as in the case of gene therapy, the intent is to have a long-lasting effect through introduction of desired genes through viral vectors, or suppression of desired genes through mechnanisms such as RNA interference. In other instances, gene therapy can lead to the removal or repair of a dysfunctional gene through use of nuclease systems such as CRISPR/Cas-9 as is being attempted with CCR5 disruption in HIV. ²⁵

A particular difficulty in developing RNA-based therapies lies in the ability to deliver it to the target cells, in part, because the highly negative charge of siRNA or miRNA makes it difficult to transport across cell membranes. Several groups are developing a variety of methods including lipid particle-based delivery modalities that are able to overcome this problem, ²⁶ though cell-specificity continues to pose a challenge to drug delivery in general. Interestingly, some strategies involve targeting alternative gene isoforms, usually for genetic diseases such as congenital muscular dystrophy or others, ^27,28 and as a result this creates a substantial opportunity for innovation, not only in the development of therapies for HIV but also for how RNA-targeting therapies are implemented in general.

Current therapeutic approaches tend to be keenly focused on the disease agent and its immediate target, for instance focusing on CD4⁺ T cells in the case of HIV. Although it is certainly correct to focus on CD4⁺ T cells since HIV preferentially targets this immune subset leading to their depletion, a disease state such as HIV infection has systemic consequences. As a result, to develop a more appropriate response, we need to consider these broader consequences of viral infection. With such readily available access to RNA-sequencing technologies, it is now possible to investigate longitudinal effects of SIV/HIV infection in a broad range of immune cells and tissues.

Current practice for publishing studies with large genomic data sets often involves deposition of these data sets in publicly accessible databases. However, most of the currently available RNA-Seq data sets on repositories such as GEO, for instance, are missing the crucial dimension of preferential gene isoform expression, in part, because of how the studies were designed. Despite this, although not all RNA-Seq data sets will provide adequate sequencing depth, it may still be informative to revisit these vast repositories to glean insights into alternative gene splicing.

Including appropriate methodologies such as direct RNA-sequencing technologies or deep-sequencing technologies from Illumina or 10x Genomics ²⁹ to quantify alternative splicing in various cell types, tissues, and in longitudinal monitoring throughout HIV/SIV infection may inform on how alternative splicing is influenced by disease state and interventions in humans and other animal models. Furthermore, understanding how alternative splicing is modulated in crucial early responding cells, such as NK cells, may provide us with a more comprehensive understanding of how HIV infection is modulating the global immune response. Perhaps HIV proteins such as TAT may also play a direct role in modulating gene splicing in NK cell since TAT has been shown to be secreted from infected cells ^30,31 or perhaps yet to be discovered immune disruptions caused by CD4⁺ T cell targeting by HIV lead to indirect NK modulations. Understanding modulation of alternative splicing during HIV infection and developing RNA-based interventions to target relevant alternative transcripts may provide us with a novel approach to treat and ultimately cure HIV/AIDS.